This year we are still participating in the many projects relating to the analysis of peptides and proteins that have been brought to our attention by researchers at NHLBI and NIH. The goal is still to use advanced mass spectrometric technology to study the biology of a particular system by obtaining information on the identity of proteins either through mass spectrometric sequencing or by simply carefully measuring the masses of a sufficient number of its peptides. This technique is unique in identifying post-translational modifications (PTMs) essential for protein/cellular functions. With the state-of-the-art Micromass QTOF3 mass spectrometer and other LC-MS and MALDI-TOF instruments, we were able to perform many experiments on biological samples, mostly in this protein and peptide class. In addition,we seek new chemical methods to enhance the search routines in order to increase their reliability. This year, we comp,leted and published on a new reagent that greatly enhances the a1 ion obtained by MS/MS analysis and proved, as estimated last year, that it is possible to locate proteins by using only the two bits of information provided by this mass and the parent ion. Studies have been initiated on the use of nitrites as derivitising reagents for peptide analysis. We have published on the identification of all of the sites of glycosylation on both human and mouse zona pellucida as well as its N- and C- terminal amino acids along with its disulfide linkages.This year we have extended this study to human ZP4 protein. We have also published on a new processing of the important stromal-derived factor 1 alpha and beta by human plasma as well as several factors involved in the specific regulation of the adaptor protein complex AP-3 by ARF GAP protein AGAP1 (P. Randazzo, NCI) using our cross linking methods worked out two years ago. We are studying a compound with activity in reducing multidrug resistance in chemotherapy and have shown that its structure is not as supposed (J. Ludwig, M. Gottesman). We have examined the structures of proteins whose cysteine groups react with peroxide and identified the location of oxidation (S.G. Rhee, NHLBI). We continue to spend a significant amount of time optimizing sensitivity and resolution of our QTOF3,its capillary LC and its communication with the mass spectrometer. The APCI method is being reexamined for its potential in analysis of peptides. A new automatic calibration device for the electrospray system has been evaluated. The new 2D-LC ProteomeX system from ThermoFinnigan is still under investigation as a source of high throughput analysis.